Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen

Metastatic melanoma is poorly responsive to treatment, and immunotherapeutic approaches are potentially beneficial. Predictors of clinical response are needed to identify suitable patients. We sought factors associated with melanoma-specific clinical response following intradermal vaccination with autologous melanoma peptide and particulate hepatitis B antigen (HBsAg)-exposed immature monocyte-derived dendritic cells (MDDC). Nineteen patients with metastatic melanoma received a maximum of 8, 2-weekly vaccinations of DC, exposed to HBsAg in addition to autologous melanoma peptides. A further 3 patients received an otherwise identical vaccine that did not include HBsAg. Patients were assessed 1-2 monthly for safety, disease volume, and cellular responses to HBsAg and melanoma peptide. There was no significant toxicity. Of 19 patients receiving HBsAg-exposed DC, 9 primed or boosted a cellular response to HBsAg, and 10 showed no HBsAg response. HBsAg-specific responses were associated with in vitro T cell responses to melanoma peptides and to phytohemagglutinin (PHA). Zero out of 10 non-HBsAg-responding and 4/9 HBsAg-responding patients achieved objective melanoma-specific clinical responses or disease stabilization- 1 complete and 2 partial responses and I case of stable disease (P=0.018). Development of melanoma-specific cellular immunity and T cell responsiveness to mitogen were greater in the group of patients responding to HBsAg. Therefore stimulation of an immune response to nominal particulate antigen was necessary when presented by melanoma peptide-exposed immature DC, to achieve clinical responses in metastatic melanoma. Since general immune competence may be a determinant of treatment response, it should be assessed in future trials on DC immunotherapy.

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4(+) T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9X10(6) or 5X10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 3 5 months +), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response (P = 0.05) and survival (log rank P < 0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.

Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer

The development of colorectal cancer is a major complication for patients with chronic idiopathic colitis. Colitis-associated tumours tend to occur at a younger age and be more aggressive than sporadic colorectal cancers. While we have previously associated the presence of tumour-infiltrating lymphocytes (TILs) and increased apoptosis in sporadic colorectal cancer with high-level microsatellite instability and improved prognosis, little is known of the relationship between these variables in colitis-associated colorectal cancer. The aim of this study was to correlate TILs and tumour cell apoptosis in colitis-associated neoplasms stratified according to microsatellite instability. Twenty tumour and 11 dysplastic samples resected from 21 patients with long-standing colitis were analysed for microsatellite instability at 10 microsatellite markers. TIL distribution (CD3, CD8) and function (granzyme B) were quantified by immunohistochemistry. Neoplastic cell apoptosis was assessed using the M30 CytoDEATH antibody. These findings were compared with 40 microsatellite stable (MSS) sporadic colorectal cancers previously evaluated for TILs and neoplastic apoptosis. Low-level microsatellite instability was found in 1/20 colitis-associated tumours. All other colitis-associated lesions were designated MSS. CD3(+) and CD8(+) TIL counts were significantly higher in colitis-associated lesions compared with NISS sporadic colorectal cancer (p < 0.0001, p = 0.001 respectively). Despite their higher TIL density, colitis-associated tumours were more likely to present late (Dukes' stage C or D) (P = 0.02). Functionally, colitis-associated TILs demonstrated significantly less granzyme B expression compared to sporadic cancers (p = 0.002). The level of tumour cell apoptosis was similar between the two groups (sporadic, 1.53%; colitis cancers, 1.45%). In conclusion, NISS colitis-associated tumours have a higher prevalence of CD3(+)/CD8(+) TILs but no associated increase in tumour cell killing by apoptosis. Unlike cytotoxic T cells in sporadic colorectal cancer, TILs do not appear to enhance the prognosis of colitis-associated colorectal cancer. This may be related to an impairment of granzyme B expression within these lesions. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the TTG in the attB1 site (30bp upstream of the ATG in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes. (c) 2006 Elsevier Inc. All rights reserved.

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4(+) T cell responses to human cytonnegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4(+) CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HICMV isolates revealed that the gB and gH epitope sequences recognized by human CD4(+) T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4(+) CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse pepticle-sensitized Patr-DR7(+) cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class 11 lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.

Histone deacetylase inhibitors specifically kill nonproliferating tumour cells

Conventional chemotherapeutic drugs target proliferating cells, relying on often small differences in drug sensitivity of tumour cells compared to normal tissue to deliver a therapeutic benefit. Consequently, they have significant limiting toxicities and greatly reduced efficacy against nonproliferating compared to rapidly proliferating tumour cells. This lack of selectivity and inability to kill nonproliferating cells that exist in tumours with a low mitotic index are major failings of these drugs. A relatively new class of anticancer drugs, the histone deacetylase inhibitors (HDI), are selectively cytotoxic, killing tumour and immortalized cells but normal tissue appears resistant. Treatment of tumour cells with these drugs causes both G1 phase cell cycle arrest correlated with increase p21 expression, and cell death, but even the G1 arrested cells died although the onset of death was delayed. We have extended these observations using cells that were stably arrested by either serum starvation or expression of the cyclin-dependent kinase inhibitor p16(ink4a). We report that histone deacetylase inhibitors have similar cytotoxicity towards both proliferating and arrested tumour and immortalized cells, although the onset of apoptosis is delayed by 24 h in the arrested cells. Both proliferating and arrested normal cells are unaffected by HDI treatment. Thus, the histone deacetylase inhibitors are a class of anticancer drugs that have the desirable features of being tumour-selective cytotoxic drugs that are equally effective in killing proliferating and nonproliferating tumour cells and immortalized cells. These drugs have enormous potential for the treatment of not only rapidly proliferating tumours, but tumours with a low mitotic index.

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting! functional rumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable. (C) 2004 Elsevier Ltd. All rights reserved.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

The efficient in vitro expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8(+) cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

HIV LTR-dependent expression of Bax selectively induces apoptosis in Tat-positive cells

HIV integrates into the host cell genome where it persists for the life of the cell. One approach to reducing viral burden is to selectively eliminate cells containing integrated provirus early following infection. We have used the HIV LTR promoter to selectively express transgenes in human cells positive for the HIV transactivator protein Tat. Transient transfection of Jurkat cells, or Jurkat cells stably expressing Tat (Jurkat-Tat), with a LTR construct containing luciferase reporter gene resulted in a 37-fold increase in gene expression when Tat was present. We have demonstrated that when pro-apoptotic Bax was used as the transgene, cytotoxicity was seen only in the Jurkat-Tat cells. Annexin-V staining indicated that Bax induced cell death by apoptosis. In mixed populations of Jurkat and Jurkat-Tat cells, the LTR-Bax construct was selectively cytotoxic to the Tat-positive cells. These results suggest that Bax under the control of the HIV LTR can be used to destroy cells harbouring HIV without affecting uninfected cells. (C) 2004 Published by Elsevier Inc.

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocircullate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HILA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathollogy in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytolkines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta 1 and alpha 5 integrins and major histocompatibility complex I molecules. The level of GIP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP(1) expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GIP-expressing cells with GIP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

RNA Loading of Leukemic Antigens into Cord Blood–Derived Dendritic Cells for Immunotherapy

The manipulation of dendritic cells (DCs) ex vivo to present tumor-associated antigens for the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTLs) attempts to exploit these cells’ pivotal role in immunity. However, significant improvements are needed if this approach is to have wider clinical application. We optimized a gene delivery protocol via electroporation for cord blood (CB) CD34+ DCs using in vitro–transcribed (IVT) mRNA. We achieved > 90% transfection of DCs with IVT-enhanced green fluorescent protein mRNA with > 90% viability. Electroporation of IVT-mRNA up-regulated DC costimulatory molecules. DC processing and presentation of mRNA-encoded proteins, as major histocompatibility complex/peptide complexes, was established by CTL assays using transfected DCs as targets. Along with this, we also generated specific antileukemic CTLs using DCs electroporated with total RNA from the Nalm-6 leukemic cell line and an acute lymphocytic leukemia xenograft. This significant improvement in DC transfection represents an important step forward in the development of immunotherapy protocols for the treatment of malignancy.